Part:BBa_K952003

B0034-YF1-B0034-FixJ-PfixK2-amilGFP

This part is a composite of BBa_K592016, BBa_K592006, and BBa_K592010 in that order. When the cells are exposed to blue-light, they will express amilGFP, a yellow chromoprotein. This protein causes the cells to have a strong yellow color.

BBa_K952003 B0034-YF1-B0034-FixJ-PfixK2-amilGFP

In the modified bacterial cells, the expression of amilgfp gene is controlled by light. We designed two sets of experiments, one with light and one with no light, to study the effect of light on amilgfp gene expression.

1. We transformed the plasmid contanining BBa_K952003-amilGFP into bacterial competent cells, plated and cultured overnight at 37 °C.

2. On the evening of the next day, we picked a single clone into a tube containing 5 ml LB and cultured overnight at 37 ° C, 220 rpm. These bacteria grow out as seed liquid. One set of tubes was incubated under light, called a light seed solution. Another set of tubes was cultured under dark conditions (wrapped with tin foil) called a dark seed solution.

3. On the third morning, we took 5ul of seed solution and inoculated it into a test tube containing 5ml of LB, which is called 0h. All the tubes were cultured under dark conditions. After 2 h, one set of tubes were continued to be darkly cultured, and the other set of tubes were stripped of tin foil for light culture. The sampling time points are 2h and 4h.

4. We took 100ul of the bacteria culture and measured the total fluorescence with a Thermo fluorescence microplate reader. We took 200ul of the ten-fold diluted bacterial solution and measured the OD600 with BioTek optical microplate reader. Total fluorescence is divided by OD600 to obtain fluorescence value per OD600.

Figure1. BBa_K952003 containing strain OD600 at 24h and 30h under dark and light.

We measured the OD600 of the strain at 2h and 4h as well as under dark and light conditions. The OD600 of the strains at 2h and 4h was 0.53 and 2.13 under dark conditions, The OD600 of the strains at 2h and 4h was 0.49 and 1.69 under light conditions. At the same time, the OD600 under dark conditions is higher than the OD600 under light conditions.

Figure2. BBa_K952003 containing strain Fluorescence per OD600 at 24h and 30h under dark and light.

We measured the RFU/OD600 of the strain at 2h and 4h as well as under dark and light conditions. The RFU/OD600 of the strains at 2h and 4h was 10.39 and 5.58 under dark conditions, The RFU/OD600 of the strains at 2h and 4h was 3.03 and 1.36 under light conditions. At the same time, the RFU/OD600 under dark conditions is higher than the OD600 under light conditions.

These results indicate that during the growth of the strain, more fluorescence is produced under dark conditions, that is, light inhibits the expression of the amlagfp gene in the strain.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 562
  Illegal NgoMIV site found at 634
  Illegal NgoMIV site found at 724
  Illegal NgoMIV site found at 742
  Illegal NgoMIV site found at 1254
  Illegal NgoMIV site found at 1547
  Illegal NgoMIV site found at 1641
  Illegal AgeI site found at 276
  Illegal AgeI site found at 1422
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1311
  Illegal BsaI.rc site found at 175